Phytofabrication of silver nanoparticles using callus extracts of natural tetraploid Trifolium pratense L. and its bioactivities

Abstract

One of the main subjects of plant biotechnology is plant tissue culture and in recent years is considered a possible approach model for green and eco-friendly biosynthesis of nanoparticles. This study aimed to present calli produced from the natural tetraploid Trifolium pratense L. containing high amounts of phenolic compounds and glycosidic bioactive macromolecules and the biosynthesis of silver nanoparticles from calli. Combinatorial optimization of silver nanoparticles was achieved for the first time in this study, thanks to the stabilizing and reducing properties of hypocotyl, apical meristem, and epicotyl derived callus extracts of the natural tetraploid T. pratense L. biosynthesized nanoparticles from three different callus extracts. Callus extracts were used to create different experiments with AgNO3 at various concentrations (0.16, 0.5, 0.84, 1.18, 1.52 and 1.96 mg L-1), different temperatures (40, 50, 60, 70, 80, 90, 100°C), and different pH levels (5, 7, 10) to carry out the biosynthesis of AgNPs. Biologically synthesized AgNPs were easily monitored by color change in ultraviolet and UV-Vis spectroscopy proved to be a fast and simple method. Also, TEM, XRD, and FTIR analyses were done to characterize and confirm the formation of crystalline nanoparticles. It was determined that antibacterial activity inhibition was achieved by using the Agar-well diffusion method for antibacterial activity measurements on Gram-positive Staphylococcus aureus ATCC 25923 and Gram-negative Escherichia coli CECT 4972 bacteria. Biosynthesized AgNPs were observed in the wavelength range of 400-500 nm in the UV-VIS spectrum. TEM analysis demonstrated the size and shape of biosynthesized silver nanoparticles under different conditions. It was observed that the smaller silver nanoparticles were spherical and the larger silver nanoparticles were triangular, elliptical, and spherical shape. The XRD analysis proved the presence of Ag0 in nanoparticles and showed crystal structure for silver nanoparticles. By FTIR analysis, O-H hydroxyl groups of functional groups on the AgNP surface, H-linked OH stretching, C-H stretching, -CH stretching of -CH2 and -CH3 functional groups, C-N and carboxylate, aliphatic phosphate and primary amine stretching were expressed. Biosynthesized silver nanoparticles showed antibacterial activity against Gram-positive S. aureus ATCC 25923 bacteria, AgNP hypocotyl (1.7mm), AgNP-epicotyl (1.1mm) against Gram-negative E. coli CECT 4972 bacteria. Among the hypocotyl, apical meristem, and epicotyl callus cultures, the highest antioxidant activity was observed in the AgNPs obtained from hypocotyl-concentration experiments, with a DPPH radical activity of 52% and an ABTS radical activity of 68%. In conclusion, these findings underscore the potential of biotechnological strategies in green nanotechnology, which can be offered for developing metal nanoparticles with potential biomedicine and biotechnology applications.